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The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 12 5594-5603
Copyright © 2002 by The Endocrine Society

Other Original Article

The Effects of Gonadotropin-Releasing Hormone (GnRH) I and GnRH II on the Urokinase-Type Plasminogen Activator/Plasminogen Activator Inhibitor System in Human Extravillous Cytotrophoblasts in Vitro

Chun-Shan Chou, Hua Zhu, Eliezer Shalev, Colin D. MacCalman and Peter C. K. Leung

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5

Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, Room 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada, V6H 3V5. E-mail: peleung{at}interchange.ubc.ca.

Abstract

The regulated expression of the urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) is believed to modulate the invasive capacity of human trophoblastic cells in vitro and in vivo. To date, the factors capable of regulating the expression of uPA and PAI-1 in these cells remain poorly characterized. In these studies, we have examined the ability of the classical mammalian GnRH I and the second form of GnRH (GnRH II) to regulate uPA and PAI-1 mRNA and protein expression levels in primary cultures of human extravillous cytotrophoblasts using quantitative competitive PCR and ELISA, respectively. Both GnRH I and II increased uPA and concomitantly decreased PAI-1 mRNA and protein expression levels in our extravillous cytotrophoblast cultures in a dose- and time-dependent manner. Cetrorelix, a peptide GnRH antagonist specific for the GnRH I receptor, was capable of inhibiting the regulatory effects of GnRH I, but not GnRH II on uPA and PAI-1 expression levels in primary cell cultures. Taken together, these observations suggest that GnRH I and GnRH II may facilitate trophoblast invasion by increasing the ratio of uPA/PAI-1 expression via interactions with two distinct GnRH receptors.

IN THE HUMAN AND higher primates, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor (PAI-1) have been shown to be spatiotemporally expressed at the maternal-fetal interface during the first trimester of pregnancy (1, 2, 3). In particular, uPA and PAI-1 have been detected in the subpopulation(s) of extravillous cytotrophoblasts that invade deeply into the decidual tissues and uterine arterioles, thereby ensuring a continuous blood supply to the placenta (4). PAI-1 increases steadily during pregnancy (5). The production of uPA by human trophoblasts is down-regulated during the second trimester, paralleling the decline in the invasiveness of these cells with gestational age. Taken together, these observations suggest that the balance between uPA and PAI-1 expression regulate, at least in part, the invasive capacity of human extravillous cytotrophoblasts. However, unlike tumor cell invasion, trophoblast invasion into the underlying maternal tissues is highly regulated (6). To date, the factors capable of regulating uPA/PAI-1 expression in highly invasive extravillous cytotrophoblasts remain poorly characterized.

GnRH is a decapeptide best known for its role in regulating the release of gonadotropins from the pituitary. However, there is increasing evidence to suggest that in addition to this classical pathway, GnRH may have direct regulatory actions on the development and function of the gonads and other reproductive tissues, particularly the endometrium and placenta (7, 8). Furthermore, recent studies have demonstrated that a distinct gene encoding a second form of GnRH, termed GnRH II, to distinguish it from the classical mammalian form (GnRH I), is expressed in the extrapituitary tissues of the human and other primates (9, 10, 11). To date, the biological function of GnRH II in the human is not known.

GnRH analogs have been shown to be capable of down-regulating the invasive capacity of breast, prostate, and uterine carcinoma cells and benign endometriotic cells in vivo and in vitro (12). These regulatory effects are believed to be mediated by the differential expression of matrix metalloproteases (MMPs), their tissue-specific inhibitors (TIMPs), uPA, and/or PAI-1. As the levels of GnRH I in the human placenta progressively increase during the first 24 wk of gestation (13), it is tempting to speculate that the invasive capacity of extravillous cytotrophoblasts may be regulated, at least in part, by the GnRH-mediated expression of uPA/PAI-1 in these cells.

Materials and Methods

Tissues and cell isolation

Samples of first trimester placental tissues were obtained from women undergoing elective termination of pregnancy (gestational ages ranging from 6–12 wk). The use of these tissues was approved by the Committee for Ethical Review of Research on the use of human subjects, University of British Columbia. All women provided informed written consent.

Extravillous cytotrophoblasts (EVTs) were propagated from first trimester placental explants as described by Graham et al. (14). Briefly, chorionic villi were washed thoroughly in DMEM (Life Technologies, Inc., Burlington, Ontario, Canada) containing antibiotics. The villi were minced finely and plated in 25-cm2 tissue culture flasks containing DMEM supplemented with antibiotics and 10% heated-inactivated fetal calf serum. The fragments of chorionic villi were allowed to adhere for 2–3 d, after which the nonadherent material was removed. The villous explants were cultured for a further 10–14 d with the culture medium being replaced every 48 h. The EVTs were separated from the villous explants by a brief (2–3 min) trypsin digestion [0.125% (vol/vol) trypsin-EDTA/Ca2+-, Mg2+-free PBS] at 37 C and plated in 60-mm culture dishes (Falcon, Becton Dickinson and Co. Labware, Franklin Lakes, NJ) penicillin/streptomycin (100 IU/ml, 100 µg/ml, respectively) and supplemented with 10% fetal bovine serum (Life Technologies, Inc.). The purity of the EVT cultures was determined by immunostaining with a monoclonal antibody directed against cytokeratin 8 and 18 (Becton Dickinson and Co.) according to the methods of MacCalman et al. (15). Only cell cultures that exhibited 100% immunostaining for cytokertain were included in these studies.

All studies were performed using EVTs (passage 2) plated in 60-mm culture plates at a density of 1 x 106 cells (Falcon, Becton Dickinson and Co.). Twenty-four hours before each treatment, serum was removed from the culture medium.

Cell treatments

To determine the effects of GnRH I or GnRH II on uPA and PAI-1 mRNA and protein levels in EVTs, cells were cultured in the presence or absence of a fixed concentration (100 nM) of GnRH I or GnRH II (Peninsula Laboratories, Inc., Belmont, CA) for 0, 3, 6, 12, 24, or 48 h or increasing concentrations of these two hormones (0, 0.1, 1, 10, or100 nM) for 24 h. The concentration of GnRH I and GnRH II used in these experiments were selected on the basis of previous studies (16, 17). In addition, EVT cultures were treated with GnRH I or GnRH II (100 nM) in combination with increasing concentrations of the peptide GnRH antagonist, Cetrorelix (AnaSpec, Inc., San Jose, CA), (1, 10, or 100 nM) for 24 h. EVT cultures treated with vehicle alone served as control for these experiments.

The cells were harvested for RNA extraction and the conditioned culture medium collected for ELISA.

Generation of first-strand cDNA

Total RNA was prepared from the EVT cultures using a RNeasy Mini Kit (QIAGEN, Valencia, CA) using the protocol recommended by the manufacturer. The concentration of total RNA present in each of the extracts was quantified by optical densitometry (260/280 nm) using a Du-64 UV-spectrophotometer (Beckman Coulter, Inc., Fullerton, CA).

An aliquot (1 µg) of the total RNA extracts prepared from these EVT cultures was reverse transcribed into cDNA using a First Strand cDNA Synthesis Kit according to the manufacturer’s protocol (Amersham Pharmacia Biotech, Oakville, Canada).

Primer design

Two sets of primers specific for human uPA or PAI-1 were designed. Nucleotide sequences specific to human uPA and PAI-1, which also spanned different exons, were identified in the human mRNA sequences (Table 1Go) deposited in GenBank (National Center for Biotechnology Information). Forward and reverse primers corresponding to these DNA sequences (primers 1 and 2 for uPA and primers 4 and 5 for PAI-1, respectively), were synthesized at the NAPS Unit, University of British Columbia. A set of competitive reverse primers (primers 3 and 6 for uPA and PAI-1, respectively) based on the same nucleotide sequence as primers 2 and 5, but in which an additional stretch of base pairs, corresponding to short nucleotide sequence identified in the target cDNAs upstream of the binding region of the original reverse primers, were incorporated into their 5'ends (Fig. 1Go). Primers specific for the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), have been previously described (18). The primer sequences and the expected sizes of the resultant PCR products are listed in Table 1Go.


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  		Table 1. Oligonucleotide primers for extravillous cytotrophoblast mRNA amplification

 


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  		Figure 1. Representative schematic diagram summarizing the construction of a competitive PCR primer for uPA. An internal standard fragment was constructed by deletion of a 203-bp fragment from the specific target cDNA to be detected.

 
PCR

PCR was performed using template cDNA generated from the total RNA extracts prepared from the cultured EVTs and combinations of the uPA and PAI-1 specific primers. The PCR conditions were as follows: 1 min at 94 C, 1 min at 57.5 C or 56 C for PAI-1 and uPA, respectively; and 1.5 min at 72 C followed by a final extension at 72 C for 15 min. The cycles were repeated 20–35 times.

A combination of primers 1 and 2 yielded the expected PCR product of 622 bp, corresponding to uPA, whereas primers 1 and 3 generated a truncated, competitive uPA cDNA of 419 bp. Similarly, a combination of primers 4 and 5 or 4 and 6 generated uPA cDNAs of 687 bp and 464, respectively. The resultant PCR products were subcloned into the PCR II vector and subjected to DNA sequence analysis to confirm the specificity of the primers. A linear relationship between the number of PCR cycles and yield of PCR product was observed after 21 cycles for GAPDH, 27 cycles for uPA and 30 cycles for PAI-1 (data not shown).

Quantitative competitive (QC)-PCR

To determine the equivalence of target cDNA and internal standard cDNA, serial dilutions of the internal standard cDNA for uPA and PAI-1 were coamplified in the presence of target cDNA. The point at which the two graphs cross indicated the amount of internal standard cDNA that should be added. One picogram of uPA or PAI-1 competitive cDNA was added to each unknown sample before QC-PCR according to the equivalences (Fig. 2Go, A and B).



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  		Figure 2. Standard curves generated for uPA and PAI. Photomicrograph of an ethidium bromide-stained gel containing uPA or PAI-1 specific PCR products generated by the coamplification of a fixed amount of target cDNA (1 µl) and serial dilutions of concentrations of competitive cDNA (8, 4, 2, 1, 0.5, 0.25, 0.125 pg/µl) (A and B, upper panel). The two lines cross in the range of 0.5–1 pg/µl and 1 pg/µl internal standard cDNA added for uPA and PAI-1, indicating that approximated 1 pg uPA and PAI-1 cNDA could be detected after RT of 1 µg total RNA (A and B, lower panel). Increasing amounts of target cDNA were coamplified with 1 pg/µl competitive cDNA. The intensities of the ratio of target and competitive cDNA generated from these reaction mixtures were determined (C and D, upper panel). The log ratios of target competitive product density plotted against the log amount of target initially added to the PCR reactions are shown in the graphs below (C and D).

 
The standard curve for uPA and PAI-1 was constructed by coamplification of a fixed amount of mutant cDNA and the varying concentration of the native cDNA obtained by serial dilutions. One microliter each of the mutant and native cDNA mix were added to 23 µl PCR-Mastermix containing 1.5 mmol/liter MgCl2 solution, 10x PCR buffer minus Mg+2, 0.2 mmol/liter of each deoxy-nucleoside 5'-triphosphate, 2.5 U Taq polymerase (all from Life Technologies, Inc.), and their corresponding paired primers (2 µmol/liter) to give a total volume 25 µl. The PCR conditions and number of cycles were performed for the uPA and PAI primer sets as described above. An aliquot (10 µl) of the PCR products was separated in a 1% agarose gel by electrophoresis and stained with ethidium bromide. The intensity of the ethidium bromide staining of the PCR products was analyzed using UV densitometry (Biometra, Whiteman Co., Gottingen, Germany). Volume counts (square millimeters) of the PCR products were then determined using the Scion Image software (Scion Image Corp., Frederick, MD). Two standard curves were then generated using the logarithmically transformed ratios of target:competitive cDNA plotted against the log amount of target cDNA included in the PCR reactions (Fig. 3Go, C and D). These standard curves were highly reproducible and linear. The values obtained from this regression curve were then used to determine the amounts of uPA or PAI-1 transcripts present in unknown samples.



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  		Figure 3. QC-PCR analysis of the effects of GnRH I or GnRH II on uPA and PAI mRNA levels in EVTs. Time-dependent effects on uPA (A and B) and PAI-1 (C and D) were determined by culturing the cells in the presence or absence of GnRH I (A and C) or GnRH II (B and D) for 0–48 h (lanes 1–6, respectively). Representative photomicrographs of the corresponding ethidium-stained gels are shown in the upper panels. A 100-bp DNA ladder is shown in lane M with the size of the target and competitive cDNAs indicated on the left. The gels were analyzed using UV densitometry. Data are presented as the means of five individual experiments and are presented as the mean ± SE (a, P < 0.001 vs. untreated control; b, P < 0.05 vs. untreated control) in the bar graphs shown in the lower panels.

 
An aliquot (1 pg) of either uPA or PAI-1 competitive cDNA was added to each unknown sample before QC-PCR was performed using the corresponding primer sets and PCR conditions described above. The ratios of the densities of sample target cDNA bands (622 and 687 bp) to competitive cDNA (419 and 464 bp) were normalized by GAPDH, logarithmically transformed and compared with the values obtained from the standard curve.

ELISA

The levels of uPA and PAI-1 in conditioned medium were measured by ELISA. uPA (Chemicon International, Inc., Temecula, CA) was detected in the conditioned culture medium with a mean intraassay and interassay coefficient of variation of 4.9% and 8.2%, respectively. PAI-1 (American Diagnostica, Inc., Greenwich, CT) was detected in the conditioned medium with a mean intraassay and interassay coefficient of variation of 6.1% and 8.8%, respectively. All samples were assayed in duplicate.

Statistical analysis

The absorbance values obtained from the ethidium bromide-stained gels were subjected to statistical analysis using GraphPad Software, Inc. Prism 2 software (San Diego, CA). Statistical differences between the absorbance values were assessed by the ANOVA. Differences were considered significant for P < 0.05. Significant differences between the means were determined using Dunnett’s test. The results are presented as the mean relative absorbance (±SEM) obtained using five or more different tissue samples.

Results

Time effects of GnRH I or GnRH II on uPA and PAI-1 mRNA levels in cultured EVT

uPA and PAI-1 mRNA transcripts were detected in all of the total RNA extracts prepared from the EVT cultures. The addition of vehicle alone had no significant effect on uPA and PAI-1 mRNA levels in cultured EVTS at any of the time points examined in these studies (data not shown).

There was a significant increase in uPA mRNA levels in EVTs with time in culture in the presence of either GnRH I or GnRH II, with maximum levels being observed after 12 h (Fig. 3Go, A and B). There was a subsequent decline in levels of the uPA mRNA transcripts in the EVTs treated with GnRH I. However, the levels were still significantly greater than those observed in the 0 h control (Fig. 3AGo). In contrast, the levels of the uPA mRNA transcript in EVTs cultured in the presence of GnRH II remained relatively constant until the termination of these studies at 48 h (Fig. 3BGo).

A significant decrease in PAI-1 mRNA levels was observed in cells cultured in the presence of GnRH II for 3 h (Fig. 3DGo). A decline in the levels of PAI-1 mRNA transcripts in EVTs cultured in the presence of GnRH I was not observed until 24 h (Fig. 3CGo). The PAI-1 mRNA levels in these cultures continued to decrease until the termination of these studies at 48 h (Fig. 3Go, C and D).

Effects of GnRH I and GnRH II of uPA mRNA and protein expression levels in cultured EVTs

GnRH I and GnRH II were capable of increasing uPA mRNA levels in EVTs in a dose-dependent manner. However, significant increases in uPA mRNA levels were only observed in EVTs treated with the higher concentrations of GnRH I (100 nM) and GnRH II (10 and 100 nM) used in these studies (Fig. 4Go, A and B).



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  		Figure 4. Effects of GnRH I and GnRH II of uPA mRNA and protein expression levels in cultured EVTs. Dose-dependent effects were determined by culturing the cells in increasing concentrations (0–100 nM; lanes 1–5, respectively) of GnRH I (A) or GnRH II (B) for 24 h. Representative photomicrographs of the corresponding ethidium-stained gels are shown in the upper panels. A 100-bp DNA ladder is shown in lane M with the size of the expected PCR products indicated on the left. ELISA analysis of uPA expression level in conditioned medium of isolated EVTs cultured in the presence of an increasing concentration of GnRH I (C) or GnRH II (D). One milligram of protein from conditioned medium was used in each ELISA. Data are shown as the means of five individual experiments and are presented as the mean ± SE (a, P < 0.001 vs. untreated control) in the bar graphs shown in the lower panels.

 
In agreement with the results obtained using QC-PCR, GnRH I and GnRH II increased uPA protein expression levels in EVT cultures in a dose-dependent manner (Fig. 4Go, C and D).

GnRH I and GnRH II decrease PAI-1 mRNA and protein expression levels in cultured EVTs

GnRH I and GnRH II decreased PAI-1 mRNA levels in a dose-dependent manner (Fig. 5Go, A and B).



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  		Figure 5. Effects of GnRH I and GnRH II of PAI-1 mRNA and protein expression levels in cultured EVTs. QC-PCR analysis of PAI-1 mRNA levels were determined by culturing the cells in increasing concentrations (0–100 nM; lanes 1–5, respectively) of GnRH I (A) or GnRH II (B) for 24 h. Representative photomicrographs of the corresponding ethidium-stained gels are shown in the upper panels. A 100-bp DNA ladder is shown in lane M with the size of the expected PCR products indicated on the left. The blots were analyzed using UV densitometry. ELISA analysis of PAI-1 expression level in conditioned medium of isolated EVTs cultured in the presence of an increasing concentration of GnRH I (C) or GnRH II (D). One milligram of protein from conditioned medium was used in each ELISA. Data are shown as the means of five individual experiments and are presented as the mean ± SE (a, P < 0.001 vs. untreated control. b, P < 0.05 vs. untreated control) in the bar graphs shown in the lower panels.

 
GnRH I and GnRH II were also found to be capable of reducing PAI-1 protein expression levels in primary cultures of EVTs in a dose-dependent manner (Fig. 5Go, C and D).

Effects of Cetrorelix on the GnRH I- or GnRH II-mediated regulation of EVT uPA and PAI-1 mRNA and protein expression levels

Cetrorelix decreased the stimulatory effects of GnRH I on uPA mRNA and protein levels in primary cultures of EVTs in a dose-dependent manner (Fig. 6Go, A and C). In contrast, the ability of GnRH II to increase uPA mRNA and protein expression was not significantly inhibited by any of the concentrations of Cetrorelix used in these studies (Fig. 6Go, B and D).



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  		Figure 6. Effects of Cetrorelix on the GnRH I- or GnRH II-mediated regulation of EVT uPA mRNA and protein expression levels. QC-PCR analysis of the EVTs. Cells were cultured in the presence of 100 nM GnRH I (A) or GnRH II (B) and an increasing amount of Cetrorelix. Representative photomicrographs of the corresponding ethidium stained gels are shown in the upper panels. A 100-bp DNA ladder is shown in lane M with the size of the target and competitive cDNAs indicated on the left hand side. The blots were analyzed using UV densitometry. ELISA analysis of uPA expression level in conditioned medium of isolated EVT cultured in the presence of an increasing concentration of GnRH I (C) or GnRH II (D). One milligram of protein from the conditioned medium was loaded in each ELISA reaction. Data are shown as the means of five individual experiments, and are presented as the mean ± SE (a, P < 0.001 vs. 100 nM GnRH, b, P < 0.05 vs. 100 nM GnRH) in the bar graphs shown in the lower panels.

 
Similarly, the inhibitory effects of GnRH I on PAI-1 mRNA and protein levels in EVT cultures were significantly attenuated in cells cultured in the in the presence of Cetrorelix (100 nM) (Fig. 7Go, A and C), whereas the addition of this GnRH antagonist to the culture medium had no effect on the down-regulation of PAI-1 mRNA and protein levels mediated by GnRH II (Fig. 7Go, B and D).



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  		Figure 7. Effects of Cetrorelix on the GnRH I- or GnRH II-mediated regulation of EVT PAI-1 mRNA and protein expression levels. QC-PCR analysis of EVTs. Cells were cultured in the presence of 100 nM GnRH I (A) or GnRH II (B) and an increasing amount of Cetrorelix. Representative photomicrographs of the corresponding ethidium stained gels are shown in the upper panels. A 100-bp DNA ladder is shown in lane M with the size of the target and competitive cDNAs indicated on the left. The blots were analyzed using UV densitometry. ELISA analysis of PAI-1 expression level in conditioned medium of isolated EVT cultured in the presence of GnRH I (C) or GnRH II (D) and an increasing amount of Cetrorelix. One milligram of protein from the conditioned medium was loaded in each ELISA reaction. Data are shown as the means of five individual experiments, and are presented as the mean ± SE (a, P < 0.001 vs. 100 nM GnRH, b, P < 0.05 vs.100 nM GnRH) in the bar graphs shown in the lower panels.

 
The ratio of uPA: PAI-1 in conditioned medium of cultured EVT treated with GnRH I or GnRH II

The ratio of uPA/PAI-1 protein levels in conditioned medium of cultured EVT in the presence of increasing concentrations of GnRH I or GnRH II was calculated. The ratio of uPA/PAI-1 in conditioned medium obtained from EVTs cultured in the presence of GnRH II was significantly greater than that obtained from cells treated with GnRH I at all of the concentrations tested (Fig. 8Go).



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  		Figure 8. Line graph depicting the ratio of uPA/PAI-1 expression levels in conditioned medium of EVTs cultured in the presence of increasing concentrations of GnRH I or GnRH II (0–100 nM).

 
Discussion

In the present studies, we have determined that GnRH I and GnRH II increased uPA-1 and concomitantly decreased PAI-1 mRNA and protein expression levels in primary cultures of EVTs, propagated from explants of first trimester chorionic villi, in a dose- and time-dependent manner. In addition, Cetrorelix was capable of inhibiting the effects of GnRH 1 but not GnRH II on uPA and PAI-1 in these cells.

GnRH I and GnRH II have been shown to elicit many diverse biological actions in extrapituitary tissues and cells. For example, GnRH modulates basal and gonadotropin-stimulated steroidogenesis (19, 20) and induces transcription of several genes involved in follicular maturation and ovulation in the ovary (21, 22). Furthermore, GnRH I and its synthetic analogs have been shown to inhibit cellular proliferation and induce apoptosis in carcinomas of the ovary (23, 24, 25). Earlier studies demonstrated that breast, ovarian, and endometrial cancers express receptors for GnRH (26, 27, 28, 29). Data available today suggest that about 50% of breast cancers and approximately 80% of ovarian and endometrial cancers express high-affinity binding sites for GnRH (30). GnRH II was found to suppress tumor cell growth in vitro (31). In addition, an autocrine/paracrine function of GnRH has been suggested to exist in the placenta (32, 33, 34, 35), granulosa cells (36, 37), myometrium (38), and lymphoid cells (39, 40, 41). For example, GnRH may act as a luteolytic agent in the ovary during the regression of the corpus luteum (42). Furthermore, in addition to the ability of GnRH I and GnRH II to regulate human chorionic gonadotropin production in the human placenta (43), our studies indicate that these two hormones may also modulate the invasive capacity of human trophoblast in vitro.

Plasminogen activators and their inhibitors (PAIs) have been identified in placenta are considered to be key participants in the balance of proteolytic and antiproteolytic activities that regulate extracellular matrix turnover. They are thought to be involved in various processes known to be associated with extensive tissue remodeling and cellular migration (44). Direct focal degradation of proteins involved in cell-cell and cell-matrix interactions by plasmin has been described (45). Complex control of the plasminogen activator cascade has been shown to be required for the movement and reorganization of cells and matrix in events such as trophoblast invasion.

To date, the mechanism(s) by which GnRH regulates uPA/PAI expression in villous cytotrophoblasts has not been determined. On possible mechanism is through the GnRH I-mediated increase in the transcription factor, AP-1 (46). Multiple AP-1 binding sites have been detected in the promoter regions of both the PAI and uPA human genes (47). GnRH I has also been shown to stimulate cAMP production in mixed pituitary cell cultures, suggesting a potential relationship between GnRH and this intracellular secondary signaling pathway (48, 49). An increase in intracellular cAMP concentration decreases the levels of PAI-1 expression in various cell lines (50, 51). The intracellular signaling events mediated by GnRH II remain to be elucidated.

The human placenta contains specific binding sites for GnRH I (52). Recently, mRNA transcripts encoding the GnRH I receptor have been detected in human placental tissues and trophoblastic cell cultures (53, 54, 55). Although the full-length mRNA transcript encoding the full-length human GnRH II receptor has not been characterized, the presence of GnRH II receptor immunoreactivity in the human pituitary and brain has been demonstrated (10, 11, 56). Furthermore, GnRH II receptor mRNA transcripts have been detected in the human term placenta (10). Our results show that Cetrorelix, an antagonist specific for the GnRH I receptor (11, 56), is able to significantly block the effects of GnRH I on uPA mRNA and protein levels. In contrast, the stimulatory effects of GnRH II on levels of uPA protein were not significantly reduced by this GnRH antagonist. These observations suggest that these effects are mediated by distinct receptors and that minimal cross-reactions occur between GnRH I and -II and their specific receptors in human trophoblasts.

The biological effects of GnRH II have been shown to be greater than those observed with GnRH I in extrapituitary cells. For example, GnRH II had a much greater effect on the inhibition of tumor cell proliferation than GnRH I and its agonists (12). Similarly, GnRH II was capable of increasing uPA mRNA and protein expression levels at lower doses and in shorter time interval than GnRH I in our primary cultures of extravillous cytotrophoblasts. These effects may be mediated by GnRH II binding to a specific high-affinity receptor with greater potency than GnRH I and/or GnRH II may be degraded at a slower rate compared with GnRH I (41). Receptor binding assays in COS-7 cells have demonstrated that GnRH II is highly selective for the type II receptor in nonhuman primates (11).

In summary, our findings demonstrated that both GnRH I and GnRH II are capable of up-regulating uPA and down-regulating PAI-1 in trophoblasts in vitro. Thus, the two types of GnRH produced in the placenta may facilitate invasion by virtue of increasing the ratio of uPA/PAI-1 and provide further evidence that members of GnRH family act in an autocrine or paracrine manner in extrapituitary tissues and cells.

Acknowledgments

Footnotes

This study was supported by a grant from the Canadian Institutes for Health Research (to P.C.K.L. and C.D.M.). P.C.K.L. is a distinguished scholar of the Michael Smith Foundation for Health Research. E.S. is a visiting scholar from the Technion Israel Institute of Technology (Haifa, Israel).

C.D.M. and P.C.K.L. made an equal contribution to these studies.

Abbreviations: EVT, Extravillous cytotrophoblasts; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMP, matrix metalloproteases; PAI-1, plasminogen activator inhibitor; QC, quantitative competitive; TIMP, tissue-specific inhibitors of MMPs; uPA, urokinase-type plasminogen activator.

Received June 5, 2002.

Accepted August 18, 2002.

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